Prokaryotic Ribosomes Research Articles

Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography, Mass Spectrometry, and Hierarchical Clustering

To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to approximately 5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114-133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The heteromeric >or=5-MDa pyruvate dehydrogenase complex and the 0.8-1-MDa acetyl-CoA carboxylase complex associated with uncharacterized biotin carboxyl carrier domain proteins constitute the entry point to fatty acid metabolism in leaves; we suggest that their large size relates to the need for metabolic channeling. Protein annotations and identification data are available through the Plant Proteomics Database, and mass spectrometry data are available through Proteomics Identifications database.

Nobel Prize in Chemistry 2009: When Biology Turns into Chemistry

Nobel Prize in Chemistry 2009: When Biology Turns into Chemistry

Ricin and Saporin: Plant Enzymes for the Research and the Clinics

Many plants produce enzymes with N-glycosidase activity, also known as Ribosome Inactivating Proteins. These proteins remove a specific adenine residue from the ribosomal RNA (28S in eukaryotes) inducing the block of pro- tein synthesis by inhibiting the binding of the Elongation Factor 2. Both eukaryotic and prokaryotic ribosomes (with dif- ferent sensitivity) can irreversibly be damaged by the action of these enzymes, suggesting their use as cytotoxic drugs. In fact several applications of targeted N-glycosidases have been developed (i.e. immunotoxins) for the treatment of human diseases such as leukaemia, but biotechnological development has furthermore suggested new applications of targeted N- glycosidases (i.e. Ig192-saporin) that are now used as powerful tools for cell biology research. The high number of en- zymes available and the possibility to express these proteins as recombinant products, allow to predict new formulations and applications discussed in this paper starting from the example of the model toxins ricin and saporin.

Ricin and Saporin: Plant Enzymes for the Research and the Clinics

Many plants produce enzymes with N-glycosidase activity, also known as Ribosome Inactivating Proteins. These proteins remove a specific adenine residue from the ribosomal RNA (28S in eukaryotes) inducing the block of protein synthesis by inhibiting the binding of the Elongation Factor 2. Both eukaryotic and prokaryotic ribosomes (with different sensitivity) can irreversibly be damaged by the action of these enzymes, suggesting their use as cytotoxic drugs. In fact several applications of targeted N-glycosidases have been developed (i.e. immunotoxins) for the treatment of human diseases such as leukaemia, but biotechnological development has furthermore suggested new applications of targeted N-glycosidases (i.e. Ig192-saporin) that are now used as powerful tools for cell biology research. The high number of enzymes available and the possibility to express these proteins as recombinant products, allow to predict new formulations and applications discussed in this paper starting from the example of the model toxins ricin and saporin. Keywords: N-glycosidases, Saporin, Ricin, toxins, immunotoxins

Abundant 5S rRNA-like transcripts encoded by the mitochondrial genome in amoebozoa.

5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified.

Dissecting the ribosomal inhibition mechanism of a new ketolide carrying an alkyl-aryl group at C-13 of its lactone ring

Ketolides are effective not only against macrolide-sensitive bacteria but also against some macrolide-resistant strains. Here we present data regarding a new ketolide with an alkyl-aryl side chain at C-13 of its lactone ring. It behaves as a strong inhibitor of protein synthesis in a model coupled transcription/translation system, although it does not affect the accuracy of translation. In addition, detailed kinetic analysis shows that it slowly forms a very tight, slowly reversible complex with prokaryotic ribosomes, a property that could be correlated with its superior activity compared with erythromycin against Escherichia coli both in vivo and in vitro.

10.1007/s11171-008-1012-4

The environment of tRNA 3′-terminus in the 80S ribosomal A and P sites was studied with a tRNAAsp analogue that bears a 4-thiouridine residue (s4U) attached to the 3′-terminal adenosine. The tRNAAsp analogue was obtained by in vitro T7 transcription followed by crosslinking with [32P]ps4Up and removal of the 3′-terminal phosphate. It was shown that the presence of the additional nucleotide at the 3′-end does not to hinder the codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in crosslinking of the analogue exclusively to 28S rRNA. The crosslinking was completely dependent on the presence of s4U in the tRNA analogue. Using hydrolysis of the crosslinked 28S rRNA with RNase H in the presence of deoxyoligomers complementary to various rRNA sequences, we determined that the crosslinking occurred in fragment 4302–4540 of the 28S rRNA. This fragment is evolutionarily conservative and belongs to domain V that is involved in the formation of the peptidyl transferase site in prokaryotic ribosomes. The use of reverse transcription allowed the determination of the tRNA analogue crosslinking in the P site to nucleotides U4461 and U4502, and the analogue in the A site, to nucleotides U4469 and C4507. In addition, nucleotide C4462 was crosslinked to both P site and A site-bound tRNA analogue. An analysis of the results demonstrates that environments of the tRNA 3′-termini are closely similar in both prokaryotic and eukaryotic ribosomes.

Microfluidic Devices for Time-Resolved Cryo-Electron Microscopy

The goal of time-resolved cryo-EM (TRCEM) is to determine structural models for transient functional states of large macromolecules such as ribosomes and ion channels. A challenge of TRCEM is to rapidly mix reactants and then deposit them as a thin aqueous film (<200 nm) on an electron microscope grid in a short time (milliseconds) before quenching of the reaction by plunging into cryogen (e.g., liquid ethane). Previously, we demonstrated that spraying the mixture with an air atomizer produces a sufficiently thin aqueous film when deposited onto a suitably hydrophilic grid. To achieve efficient mixing, low sample consumption (several microliters/sec), and flow rates compatible with air atomization, we designed and fabricated planar passive mixers consisting of a pair of premixers and a single channel containing pillar obstacle structures. A computational fluidic dynamics (CFD) simulation, using FLUENT CFD was used to direct our mixer designs. Experimental evaluations of mixer performance at millisecond time scales were conducted by fluorescence microscopy using fluorescent dye solutions or nanoparticle suspensions. Now we have integrated a micro-mixer and several types of sprayer into silicon-based chips, and have begun to test their feasibility for TRCEM. Initial experiments have focused on the kinetics of the assembly of prokaryotic ribosomes from their constituent 30S and 50S subunits. Assembly of ribosomes has been achieved by TRCEM, and experiments to detect potential intermediates in the process are under way.

Ethambutol Optic Neuropathy: How We Can Prevent 100,000 New Cases of Blindness Each Year

Ethambutol Optic Neuropathy: How We Can Prevent 100,000 New Cases of Blindness Each Year

The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.

Panning for chemical gold: marine bacteria as a source of new therapeutics

Marine bacteria are emerging as an exciting resource for the discovery of new classes of therapeutics. The promising anticancer clinical candidates salinosporamide A and bryostatin only hint at the incredible wealth of drug leads hidden just beneath the ocean surface. For example, if properly developed, marine bacteria could provide the drugs needed to sustain us for the next 100 years in our battle against drug-resistant infectious diseases. This review will focus on several recently discovered compounds, primarily from cyanobacteria and actinobacteria, that illustrate the tremendous potential of marine bacteria as a source of new therapeutics within the areas of oncology and infectious diseases.

Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrug-resistant bacteria. In this study, we characterized several members of a new oxazolidinone family, R chi-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the R chi-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether R chi-01 compounds protect U2585 (Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The R chi-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the R chi-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the R chi-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the R chi-01 family of compounds is currently undergoing clinical trials.

Arrangement of 3′-terminus of tRNA on the human ribosome as revealed from cross-linking data

This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3′-end of tRNA in the 80S ribosomal A and P sites using a tRNA Asp analogue that bears a 4-thiouridine (s 4U) attached to the 3′-terminal adenosine. It was shown that an additional nucleotide s 4U77 on the 3′-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302–4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s 4U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3′-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.

Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.

Pokeweed antiviral protein region Gly209–Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome

Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209–225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209–225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209–225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.

Translational Regulation via L11: Molecular Switches on the Ribosome Turned On and Off by Thiostrepton and Micrococcin

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A 51 residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T. cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.

Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.